Rotavirus (RV) infection is the leading cause of childhood diarrheal morbidity and mortality worldwide despite the recent approval of two safe and effective live attenuated vaccines. Many fundamental questions concerning the basis of heterotypic humoral immunity, the mechanisms by which RV elicits but also avoids the innate immune response, and the factors controlling tissue tropism, systemic replication, and host range restriction remain to be answered. In this proposal, we will use a series of related and complementary approaches to characterize the mechanisms by which specific components of the virus and the host immune systems interact to either enhance or restrict RV replication and pathogenesis in the host. These aims are to: 1) Define the clonal and structural basis of heterotypic humoral rotavirus immunity in humans. Heterotypic (serotype cross-reactive) immunity plays an important role in preventing RV disease and is a critical contributor to the efficacy of at least one of the two currently licensed RV vaccines. Heterotypic humoral immunity is also a key component of several established (e.g., influenza) and experimental (e.g., HIV and HCV) vaccines. We propose to define, at the individual immunoglobulin (Ig) molecule level, the serologic nature of heterotypic neutralizing reactivity and the structural basis for this reactivity. Our hypothesis is that humans circumvent the serotypic diversity of circulating RV strains by developing lg molecules directed at VP4, VP7, and VP6 that mediate heterotypic protection. 2) Identify RV genes responsible for variation between strains in their ability to replicate systemically and cause systemic disease and characterize the link between interferon (IFN) interference and cellular tropism. RV spreads and replicates systemically although strains vary substantially in both the level and sequaelae of the systemic phase of infection. IFN signaling appears to play an important role in regulating systemic replication of different RV strains. We will determine, in vivo, which RV genes are responsible for the variation in systemic replication capacity and distinct organ specificity of different RV strains and investigate how the strain-dependent effects of NSP1 on IFN signaling affect this cell tropism and host range. Our hypothesis is that the NSP1 protein (gene 5 product), through its host-specific inhibitory effects on IFN signaling, plays an important, but not exclusive role in regulating cell tropism and systemic RV replication. 3) Characterize the interaction of murine plasmacytoid dendritic cells (pDCs) with RV and determine the contribution of pDC-derived interferon to restricting RV infection. Recent studies in humans and animals demonstrated that RV infection is not limited to the mature epithelial tip cells of the small bowel but also occurs in other cells at other sites, including DCs. pDCs are a primary source of the host anti- viral IFN response and studies by us and others provide support for the notion that IFN is an important regulator of RV pathogenesis that specifically functions to restrict systemic replication. Here we will characterize the interaction between murine pDCs and RV in vitro and use an in vivo mouse model to determine the role of pDCs in restricting systemic replication as well as B cell activation. Our hypothesis is that RV interaction with pDCs plays a critical role in restricting systemic RV replication and that pDCs, unlike many other host cells, are capable of circumventing RV's inherent ability to block the induction of IFN. PUBLIC HEALTH RELEVANCE: Rotaviruses are important in their own right since they kill more than half a million children per year. They also represent a highly tractable system to study basic aspects of innate, acquired and mucosal immunity and viral pathogenesis, all important issues for the future of infectious disease prevention and treatment.